The present invention is related with the field of immunology and human medicine particularly with the generation and selection of a monoclonal antibody (Mab) against the N-glycolylated-galactose-glucose sialic acid olygosaccharide sequence that can be used for the diagnosis and treatment of certain neoplasic diseases.
The olygosaccharide structures can be found forming part of glycoproteins and glycolipids. They are both present in normal and pathological tissues.
The aberrant glycosilation has been described in approximately 100% of the malignant neoplasm. Frequent changes in the aberrant glycosilation are: the expression of neoantigens, variations in the composition of the olygosaccharide sequences, increase or decrease of the sialic acid molecules in the olygosaccharides and increase in the density of the molecules in the cell surface, among others (Hakomori S. H. et al. Curr. Opin. in Immunol. 1991,(3) 646-653). In addition to the changes that can be found in the mechanism of the sialyl-tranferases, there are also variations in the activated sialic acid N-acetylated dependent hydroxilases.
Gangliosides are glycoesfingolipids that contain sialic acid in their structure and are characterized by being present in most cells of the vertebrates. These molecules are found in normal tissue and can have a higher expression in the tumors, with a different organization and conformation in the surface of malignant cells (Hakomori, S H., 1985, Cancer Res. 45: 2405-2414; Miraldi, F., 1989, Seminars in Nuclear Medicine, XIX,282-294).
The humoral immune response against carbohydrate antigens is generally of the IgM isotype. The olygosaccharide sequences bound to lipids are generally less immunogenic than the glycoproteins. Thus, the use of glycolipids as immunogen requires of its binding to transporting proteins or their incorporation to liposomes or to bacteria such as Micobacterium tuberculosis or R595 de Salmonella minnesota. 
The response generated against Gangliosides is thymus independent. This has been reported repeatedly by Livingston, et al., (Livingston, P. O. et al., 1982, Proc. Natl. Acad. Sci. USA 84: 2911-2915; Livingston, P. O. et al. 1989, Cancer Res. 49: 7045-7050). The main characteristics of the antibodies generated against Gangliosides when studied in the serum of different species are their low affinity, considerable cross reactivity and short life (Livingston, P.O. 1991, Immunology and Allergy Clinics of North America, 11: 401-423; Portoukalian, J. et al, 1991, Int. J. Cancer, 49: 893-899).
The expression of the N-glycolylated form in the olygosaccharides is common in normal and pathological tissues of all the species of vertebrates, except for chickens and humans in which it is only found in fetal and tumoral tissue. The normal tissues of these two last species posses only the N-acetylated variant (Nishimakit et al. 1979, J. Immunology, 122: 2314; Higashi H. et al, 1985, Cancer Res., 45: 3796). 
The study of the olygosaccharide composition in some human tumors demonstrate the presence of the N-glycolylated form both in glycolipids and glycoproteins of melanoma tumoral cells (Hirabayashi, Y, et al. 1987, J. Cancer Res., 78, 614 -620; Saida T. et al. 1991 Arch. Dermatol. Res. 282(3): 179-182; Kawashima I. et al. 1993, J. Biochem (Tokio) (2) 186-193; Kawachi S. et al., 1992, J. Dermatol (11): 827-830), as well as in colon tumors, especially in glycolipids (Miyoshi, I., et al, 1986, Mol. Immunol. 23(6): 631; Higashi H., et al, 1985, Cancer Research, 45: 3796-3802). Additionally, studies have been performed to demonstrate the presence of the N-glycolylated form of the Gangliosides in tumoral samples of liver, teratoma, lymphoma, etc, (Kawai T. et al. 1991 Cancer. Res. (51) 1242-1246). Although in the former cases the concentration of the N-glycolylated variant of glycolipids was less than 0,05% of the total sialic acid, Marquina and collaborators found in breast tumors values of approximately 10% of the sialic acid bound to lipid (Marquina, G. et al, 1996, Cancer Res. 56: 5165-).
The generation of monoclonal antibodies against the N-glycolylated variant of the gangliosides has provided until now, antibodies of the IgM isotype that generally recognize more than one gangliosides molecule, for example, the human monoclonal antibodies 2-39 M and 32-27 M (Furukawa K., et al, 1988, J. Biological Chemistry, 263: 18507) and the murine antibodies GMR8 and GMR3 (Ozawa H. et al, 1992, Biochem. Biophys., 2(294):427). Other authors have reported the generation of a specific species of anti N-glycolylated gangliosides antibodies, always of IgM isotype, among which are the monoclonal antibodies Y-2-HD1, against NGcGM2 (Samai Y. et al, 1988, Bioch Biophys. Act., 958, 368) and MK2-34 against the same molecule (Miyake, M. et al, 1990, Cancer Res. 48, 6154).
Nevertheless, Watarai (Watarai, S. et al. 1995. J. Biochem. 117, 1062) generated the monoclonal antibody SHS-1 against the i-active N-glycolylated gangliosides and Nakumara obtained the monoclonal antibody YK-3 against the (NGc-NGc) GD1c (Nakumara et al, 1995, J. of Biolog. Chemist., 8 (270):3876). Recently Vxc3xa1zquez, et al., (Vxc3xa1zquez, A. M. et al, 1995, Hybridoma, 14, 6, 551) reported the generation of the monoclonal antibody P3, that recognizes most Ganglioside molecules containing the N-glycolylated form of the sialic acid, as well as the sulfated glycolipids.
Nagai et al., have generated the HMA1 monoclonal antibody against Gangliosides (Nagai Y. et al. U.S. Pat. No. 4,965,198). They obtained a specific monoclonal antibody against the Ganglioside NGcGM2 from mice bearing an autoimmune disease. Although, they reported several of these antibodies that additionally recognized other N-glycolylated Gangliosides designated as PyK, YH02, YH03, YH04, YH05, YH06 y YH07.
Moreover, Yamasaki, M. et al., in their U.S. Pat. No. 4,942,131 report the generation of the Mabs YH08, YH09, YH10 e YH11 against the 4-O-Acetyl-NGcGM3 Ganglioside, also in mice with an autoimmune disease.
Monoclonal antibodies against Gangliosides have also been obtained using these molecules as lactones or from cell lines containing Gangliosides (U.S. Pat. Nos. 5,308,614; 5,240,833; 5,389,530 y 5,500,215).
In the same manner, different monoclonal antibodies, both murine and human, have been obtained against GD3, GD2 y GM2 gangliosides, all of the N-acetylated form and most of them of the IgM and IgG3 subclasses (Pukel, C. S. et al. 1982, J. Exp. Med., 115: 1133-1147; Hirabayashi, Y. et al. 1985, J. Biol. Chem., 260: 13328-13333; Patent application WO 86/00909; Miyake, M. et al. 1988, Cancer Res., 48: 6154-6160; Kawashima, I. et al. 1992, Molecular Immunology, 29, 625-632; Kotani, M. et al. 1992, Biochimica et Biophysica Acta, 1117: 97-103).
The passive immunotherapy with monoclonal antibodies against gangliosides has been used in clinical trials for the treatment of some tumors such as melanomas and neuroblastomas. Treatment of melanomas have been intra lesion or systemic and although results seem to be encouraging, only a reduced number of patients showed total or partial remissions (Houghton, A. N. et al, 1985, Proc. Natl.Acad. Sci. USA, 82: 1242; Dippold, W. G. et al, 1988, J Cancer Clin. Oncol., 24: 865; Vadhan-Raj, S. et al. 1988, J. Clin. Oncol., 6: 1636; Saleh M. N. et al. 1992, Cancer Res., 52: 4332-4347).
These antibodies showed effect in complement or cell mediated cytotoxicity studies (Ravindramath M. H. et al. 1991, Inter. Rev. Immunol., 7, 303).
Up to now, all the monoclonal antibodies obtained against N-glycolylated gangliosides are of the IgM isotype and the toxicity they provoke is mediated by complement.
IgM""s generally have low antigen affinity and it is difficult to use them for diagnosis or treatment as radiolabeled Mabs. Although they fix complement well and guarantee a good cytotoxicity, the possibility of large-scale purification is much more complicated than with the IgG isotype.
Moreover, little has been reported on monoclonal antibodies against N-glycolylated glycoproteins, and most of them for diagnostic purposes.
Devine et al., described the 3E1.2 monoclonal antibody which recognizes an N-glycolylated mucin (glycoproteic) expressed in 90% of breast tumors studied by immunohistochemistry (Devine, P. L., et al. 1991, Cancer Res. 51(21): 5826-36).
It has also been published a monoclonal antibody designated JAM3, that recognizes the N-acetylated and N-glycolylated forms of a 250 kD protein present on the surface of the cysts produced by the attack of Entamoeba (Avron, B., et al. 1987, Mol Biochem Parasitol. (3): 257-266).
The novelty of the present invention consists in having obtained a monoclonal antibody highly specific for the N-glycolylated-galactose-glucose sialic acid olygosaccharide sequence, present in both, gangliosides and glycoproteins. Additionally the characteristic of being an IgG isotype immunoglobulin makes it more specific and thus of higher affinity for the molecules it recognizes, favoring its biological activity. Unexpectedly this antibody showed the capacity to provoke cellular death directly in the cells bearing said olygosaccharide sequence.
Obtention of the NGcGM3 Ganglioside
For obtaining the NGcGM3 ganglioside a modification of Hakomori""s technique is used. (Hakomori, S. et al. 1974, Methods in Enzymology, 32:, Part B, 350), using natural sources such as horse erythrocytes. The yield of NGcGM3 Ganglioside extraction was between 180 and 300 mg per liter of horse erythrocytes with a purity above 90% corroborated by high efficiency liquid chromatography according to, Gazzotti""s method (Gazzotti, G. et al. 1985, J. of Chromatography, 348: 371-378).
Obtention of the Immunogen
To obtain the immunogen the NGcGM3 Ganglioside is hydrophobically bound to the human lipoproteins of very low density (VLDL) obtained according to Dumontet et al., (Dumontet, C. et al. 1994. Cancer Immunol Immunother. 38: 311-318.
Immunization Scheme
To obtain anti ganglioside IgG Mabs the following immunization method is used. Mice or other mammalian species were immunized with vaccine preparations containing between 0.03 and 0.5 mg of the NGcGM3 Ganglioside bound to VLDL per dose and an adjuvant selected from one of the following: albumin, complete or incomplete Freund""s adjuvant or Montanide ISA 51.
Before and after the immunization period, blood samples are taken from the animals to obtain serum for monitoring the antibodies generated in the animals against the Ganglioside used as antigen. Any of the known immunoassay methods for detecting the antigen-antibody (Ag-Ab) reaction is used for this purpose.
The animals are immunized with various doses, between 2 and 8 at time intervals varying between 7 and 14 days. The administration is performed by subcutaneous or intramuscular route with volumes between 0.1 and 0.2 mL. Other possible immunization routes are intravenous and intraperitoneal. The animals receiving this dose range show a specific response against the Ganglioside used as immunogen. Between 70 and 100% of the immunized animals had a specific IgG response to the NGcGM3 ganglioside.
Achievement of the Monoclonal Antibodies
For the production of specific Mabs against the NGcGM3 ganglioside the mice with antibody titers in serum against this ganglioside received a new immunization with the vaccine preparation 3 days before the antibody producing cells are obtained. Spleen cells should be preferred although other cells can also be used.
These cells are fused with myeloma cells, which provide the hybrid cells or hybridomas with the capacity to expand indefinitely xe2x80x9cin vivoxe2x80x9d and xe2x80x9cin vitroxe2x80x9d. For this purpose any of the known cell fusion methods can be used. To determine the antibodies produced by the hybridomas an immunoenzymatic assay is preferentially used. Other immunoassay methods can also be used. The procedure of this assay is the recognition by hybridoma supernatans of the gangliosides, and the antigen-antibody reaction can be visualized using a second antibody labeled with an enzyme which binds to the antibody produced by the hybridoma under adequate conditions and is at the same time detected.
The hybridoma once selected is cloned at least 2 times (for example, by limiting dilution). The Mab obtained can be produced xe2x80x9cin vitroxe2x80x9d in an adequate culture media, such as any of the ones described in the state of the art and afterwards purified from said tissue culture supernatant. In this case, between 1 and 8% of the secreting clones were specific against the N-glycolyl Gm3 Ganglioside.
Another antibodies production method, consists in the injection of the hybridoma in animals (for example, syngenic animals). The hybridoma provokes the formation of non-solid tumors that provide a large concentration of the desired antibody in the blood stream and in the peritoneal exudate of the host animal.
Purification of the Monoclonal Antibody
The purification of the monoclonal antibodies is performed from the ascitic fluid obtained by the inoculation of 0.2xc3x97106 cells of the monoclonal antibody producing hybridoma in the peritoneal cavity of Balb/C mice, previously treated with incomplete Freund adjuvant as ascitogenic agent.
The ascitic fluid is diluted to one half in glycine buffer 1.5 M, NaCl, 3M, pH 8.9 and applied to a protein A-Sepharose matrix at a flow rate of 60 mL/h. The Mab elution is performed using citrate buffer 0.14 M, pH 6.
The concentration of the purified Mabs is estimated by the Lowry method (Lowry, G. H., 1951, J. Biol. Chem., 193: 256) and using the absorption coefficient of the murine IgGl at 280 nm. The specificity is confirmed by ELISA.
Between 2 and 5 mg of antibody per mL of ascitis were obtained, with purity per cent above 95%. This was corroborated by low-pressure liquid chromatography.
Specificity Studies
To determine the specificity of the monoclonal antibodies obtained, immunoenzymatic studies of the Mabs produced are performed in ELISA plates and in thin layer chromatography using the N-acetylated (GM1, GM2, GM3, GM1, GD1a, GD1b, GD3 and GT1b) and N-glycolylated (GM3, GM2, GM1a, GM1b, GD1c y GD3) Gangliosides.
To run the glycolipids in the high-resolution thin layer chromatography the solvent system is used (Chloroform:Methanol:KC1 0.25% and 2.5 M NH3) (5:4:1) (v:v). Chemical developing with Orcinol performs the visualization of the bands.
The plates are plastic coated with a poliisobutilmethacrilate solution and are air dried at room temperature over night. Blocking is performed during approximately 30 minutes with a 1% bovine serum albumin solution dissolved in saline phosphate buffer (PBS), pH 74. Afterwards, the monoclonal antibodies are incubated in the blocking solution.
Next the plates are washed with PBS and the peroxidase conjugated anti mouse immunoglobulin is added during one hour. Plates are again washed and the enzyme substrate solution is added until the bands are visualized. Finally the chemical and immunological development are compared. As a result the IgG recognized only the NGcGM3 ganglioside.
Cytotoxicity Determination
To determine if the antibodies generated produce cell death directly or by some of the other cytotoxic forms, 107 cell/mL of the P3X63 murine myeloma containing NGcGM3 is incubated at 4xc2x0 C. and 37xc2x0 C. respectively with the monoclonal antibody between 0.01 and 1 mg/mL during 30 minutes. Then the Tripan blue method is used for performing the viability studies. The number of dead cells as consequence of the antibody effect can be counted using propidium iodine or any other viability marker.
To study the complement mediated cytotoxicity 107 cells per mL were used; monoclonal antibodies are added at concentrations between 0.01 and 0.5 mg/mL. Rabbit serum that has high concentrations of the complement proteins, is added at dilutions from {fraction (1/20)} to xc2xd and incubated during 1 hour at 37xc2x0 C. Complement mediated cytotoxicity was determined by viability counts as described above or using the Cr51liberation method in which the P3X63 radiolabeled myeloma cells, liberate to the culture supernatant the isotope when they die.
The direct cytotoxicity was measured using different methods that showed values between 50 and 85% of dead cells with respect to the total number of cells studied.
Monoclonal Antibody Biodistribution Determination
The Mabs generated can be used both for diagnosis and treatment, labeled with a radioisotope such as 99mTc, Re186 and Re188. Schwarz and Steinstrasser (Schwarz A., and Steinstrasser, A. 1987, J. Nucl Med. 28: 721) have described the method of labeling monoclonal antibodies with radioisotopes which was modified by Mather y Ellison (Mather S. J. and Ellison D., 1990, J. Nucl. Med. 31: 692-697). Labeling quality control is performed by chromatography in Whatman 3MM paper. The per cent of labeling obtained was 98 and 100%.
To determine the possible use of the Mab, 10 mice were inoculated with the P3X63 tumor and another 10 mice were used as normal controls (no tumor was inoculated). The time needed for tumor to grow was waited and then the 99mTc-labeled 14F7 Mab was injected by intravenous route to the 20 mice.
Monitoring of the biodistribution of the anti- olygosaccharide sequence Mab is performed in groups of 5 animals (5 healthy and 5 with tumor) 4 and 24 hours post injection. The animals are sacrificed and the main organs and tumor are weighed and the gamma emission quantified separately at the end of the study.
The monoclonal antibodies distributed in the healthy animals mainly in the blood, liver and kidney while in the animals with tumor the Mab was localized in the former organs and preferentially in the tumor at 24 hours.
Mab""s Recognition of Normal and Fetal Tissues
Radiolabeled Mabs, as described in the state of the art, can be used to detect tumors where the olygosaccharide sequence is expressed.
Whole body radioactivity can be studied with a Gamma Camera. Images acquisition is performed at 5 minutes and 1, 3, 5, 24 and 48 hours after Mab injection. Mab is localized only in the tumor and in the excretion organs.
Mabs can also be bound directly or indirectly to other therapeutic agents such as drugs, radioisotopes, immunomodulators, lectins and toxins. Among the biological response modifiers (immunomodulators) that in some way can increase the destruction of the tumor by the Mab of this invention are included lymphokines such as: Tumor Necrosis Factor, Macrophage Activator Factor, Colony Stimulating Factor, Interferons, etc.
Immunohistochemical studies were performed for diagnostic purposes. Tissue sections were fixed in 10% buffered formaline solution and dehydrated, clarified and embedded in paraffin. Histopathology was studied in Hematoxilin-Eosin stained tissue sections.
Serial sections from the paraffin blocks used for the histopathological study were immunostained by the biotin streptavidin peroxidase complex method, previously described (Hsu, S. M. y Raine, L., 1981,. J. Histochem Cytochem. 29: 1349-1353).
The deparaffinized and dehydrated sections were treated with 3% hydrogen peroxide methanol solution during 30 minutes to eliminate endogenous peroxidase activity. Tissue sections were incubated with the purified Mabs. Followed by biotinilated anti mouse antibodies and streptavidin peroxidase complex (Dakopatts) at room temperature.
Between incubations sections were washed with Tris-HCl saline buffered solution. The peroxidase reaction was developed with 30% H2O2 and 3-3 diaminobencidine.
Slides were washed with tap water, stained with Mayer""s Hematoxilin, mounted with balsam and coverslipped. The reaction with the enzyme produces a brown-red color.
Human breast, lung, skin and nervous system tumoral tissues were studied as well as, fetal and normal adult tissues.
Fresh biopsies of pathological tissues were obtained during the first hour after surgery. The biopsy fragments were frozen, later sectioned and the slides stored frozen until the study was performed.
The use of fetal tissues for the study is due to the fact that the association of Gangliosides with oncofetal antigens has been repeatedly reported as well as the similarity of these molecules in fetal and tumoral human (Cahan, L. et al. 1982 Proc. Natl. Acad. Sci. USA., 79:7629-7633).
The fetal tissue sections were obtained from fetus between 12 and 18 weeks old.
Adult normal tissue fragments were obtained from individuals deceased in accidents and/or encephalic death during the first hour after exitus letalis.
Among the tumors studied, lung and central nervous system tumors of different ethiology resulted negative as well as the sections of normal human tissues. While melanoma and breast tumor tissue sections were all positive as well as the fetal tissue sections of the digestive system (liver, stomach, small and large intestine) and the renal system.
Antitumoral Effect
To demonstrate the anti tumoral effect of the monoclonal antibodies against the NGcGM3 Gangliosides, animals inoculated with the tumor bearing the target Ganglioside (P3X63 myeloma) was treated with the antibodies obtained. The dose can vary from 0.01 mg/kg of weight to 200 mg/kg of weight in one or more daily administrations during one or various days. The antibodies can be administered by parentheral injection (intravenous, intraperitoneal, intramuscular, subcutaneus, intracavity or transdermic).
In a typical experiment the mice treated with the antibody have a survival rate between 30 and 80% compared to with the mice of the control group, corroborated by the Log Ram test (Cox and Oakes (1984) Analysis of survival Data edits. Chapman Hall). Significant differences ( less than 0.05%) were found between the group treated with the Mab and the control group.